Autoradiographic characterization of [3H]-5-HT-moduline binding sites in rodent brain and their relationship to 5-HT1B receptors

5-HT-moduline is an endogenous tetrapeptide [Leu-Ser-Ala-Leu (LSAL)] that was first isolated from bovine brain tissue. To understand the physiological role of this tetrapeptide, we studied the localization of 5-HT-moduline binding sites in rat and mouse brains. Quantitative data obtained with a gaseous detector of β-particles (β-imager) indicated that [3H]-5-HT-moduline bound specifically to rat brain sections with high affinity (Kd = 0.77 nM and Bmax = 0.26 dpm/mm2). Using film autoradiography in parallel, we found that 5-HT-moduline binding sites were expressed in a variety of rat and mouse brain structures. In 5-HT1B receptor knock-out mice, the specific binding of [3H]-5-HT-moduline was not different from background labeling, indicating that 5-HT-moduline targets are exclusively located on the 5-HT1B receptors. Although the distribution of 5-HT-moduline binding sites was similar to that of 5-HT1B receptors, they did not overlap totally. Differences in distribution patterns were found in regions containing either high levels of 5-HT1B receptors such as globus pallidus and subiculum that were poorly labeled or in other regions such as dentate gyrus of hippocampus and cortex where the relative density of 5-HT-moduline binding sites was higher than that of 5-HT1B receptors. In conclusion, our data, based on autoradiographic localization, indicate that 5-HT-moduline targets are located on 5-HT1B receptors present both on 5-HT afferents and postsynaptic neurons. By interacting specifically with 5-HT1B receptors, this tetrapeptide may play a pivotal role in pathological states such as stress that involves the dysfunction of 5-HT neurotransmission.

Pleiotropic Alterations in Lipid Metabolism in Yeast sac1 Mutants: Relationship to “Bypass Sec14p” and Inositol Auxotrophy

SacIp dysfunction results in bypass of the requirement for phosphatidylinositol transfer protein (Sec14p) function in yeast Golgi processes. This effect is accompanied by alterations in inositol phospholipid metabolism and inositol auxotrophy. Elucidation of how sac1 mutants effect “bypass Sec14p” will provide insights into Sec14p function in vivo. We now report that, in addition to a dramatic accumulation of phosphatidylinositol-4-phosphate, sac1 mutants also exhibit a specific acceleration of phosphatidylcholine biosynthesis via the CDP-choline pathway. This phosphatidylcholine metabolic phenotype is sensitive to the two physiological challenges that abolish bypass Sec14p in sac1 strains; i.e. phospholipase D inactivation and expression of bacterial diacylglycerol (DAG) kinase. Moreover, we demonstrate that accumulation of phosphatidylinositol-4-phosphate in sac1 mutants is insufficient to effect bypass Sec14p. These data support a model in which phospholipase D activity contributes to generation of DAG that, in turn, effects bypass Sec14p. A significant fate for this DAG is consumption by the CDP-choline pathway. Finally, we determine that CDP-choline pathway activity contributes to the inositol auxotrophy of sac1 strains in a novel manner that does not involve obvious defects in transcriptional expression of the INO1 gene.

Phenotype of arylsulfatase A-deficient mice: Relationship to human metachromatic leukodystrophy

Metachromatic leukodystrophy is a lysosomal sphingolipid storage disorder caused by the deficiency of arylsulfatase A. The disease is characterized by progressive demyelination, causing various neurologic symptoms. Since no naturally occurring animal model of the disease is available, we have generated arylsulfatase A-deficient mice. Deficient animals store the sphingolipid cerebroside-3-sulfate in various neuronal and nonneuronal tissues. The storage pattern is comparable to that of affected humans, but gross defects of white matter were not observed up to the age of 2 years. A reduction of axonal cross-sectional area and an astrogliosis were observed in 1-year-old mice; activation of microglia started at 1 year and was generalized at 2 years. Purkinje cell dendrites show an altered morphology. In the acoustic ganglion numbers of neurons and myelinated fibers are severely decreased, which is accompanied by a loss of brainstem auditory-evoked potentials. Neurologic examination reveals significant impairment of neuromotor coordination.

Prospective measurements of dehydroepiandrosterone sulfate in a cohort of elderly subjects: Relationship to gender, subjective health, smoking habits, and 10-year mortality

The decrease with age of the adrenal-secreted dehydroepiandrosterone sulfate (DHEAS) in serum has suggested that it may be causally related to longevity. For the PAQUID [People (Personnes) Aged (Agées) About What (Quid, in Latin)] cohort of elderly subjects, we have previously reported higher DHEAS in men than in women, a decrease with age and, among men, a negative correlation between the DHEAS level and mortality at 2 and 4 years. Here, with an 8-year followup in 290 subjects, we show a global decrease of 2.3% per year for men and 3.9% per year for women. However, in approximately 30% of cases, there was an increase of DHEAS. We observed no relationship between the evolution of DHEAS level and functional, psychological, and mental status, possibly because of selection by death. In women, no association was found between mortality and DHEAS level. In men, the relative risk (RR) of death was higher for the lowest levels of DHEAS (RR = 1.9, P = 0.007), with RR = 6.5, P = 0.003 for those under 70 years old, a result indicating heterogeneity of the population. There was an effect of subjective health on mortality that disappeared after adjustment of DHEAS levels, suggesting its relation with these DHEAS levels. Death RR was much higher in smokers with a low DHEAS level than in nonsmokers with high DHEAS (RR = 6.7, P = 0.001). We submit that the involvement of DHEAS is possibly different according to gender, that association between low DHEAS level and mortality only for men under 70 years old possibly reflects heterogeneity of the population, and that DHEAS level is a reliable predictor of death in male smokers.

Myelin basic protein kinase activity in tomato leaves is induced systemically by wounding and increases in response to systemin and oligosaccharide elicitors

In response to wounding, a 48-kDa myelin basic protein (MBP) kinase is activated within 2 min, both locally and systemically, in leaves of young tomato plants. The activating signal is able to pass through a steam girdle on the stem, indicating that it moves through the xylem and does not require intact phloem tissue. A 48-kDa MBP kinase is also activated by the 18-amino acid polypeptide systemin, a potent wound signal for the synthesis of systemic wound response proteins (swrps). The kinase activation by systemin is strongly inhibited by a systemin analog having a Thr-17 → Ala-17 substitution, which is a powerful antagonist of systemin activation of swrp genes. A 48-kDa MBP kinase activity also increases in response to polygalacturonic acid and chitosan but not in response to jasmonic acid or phytodienoic acid. In def1, a mutant tomato line having a defective octadecanoid pathway, the 48-kDa MBP kinase is activated by wounding and systemin as in the wild-type plants. This indicates that MBP kinase functions between the perception of primary signals and the DEF1 gene product. In response to wounding, the MBP kinase is phosphorylated on phosphotyrosine residues, indicating a relationship to the mitogen-activated protein kinase family of protein kinases.

Structure of sortase, the transpeptidase that anchors proteins to the cell wall of Staphylococcus aureus

Surface proteins of Gram-positive bacteria play important roles during the pathogenesis of human infections and require sortase for anchoring to the cell-wall envelope. Sortase cleaves surface proteins at the LPXTG motif and catalyzes the formation of an amide bond between the carboxyl group of threonine (T) and the amino group of cell-wall crossbridges. The NMR structure of sortase reveals a unique β-barrel structure, in which the active-site sulfhydryl of cysteine-184 is poised for ionization by histidine-120, presumably enabling the resultant thiolate to attack the LPXTG peptide. Calcium binding near the active site stimulates catalysis, possibly by altering the conformation of a surface loop that recognizes newly translocated polypeptides. The structure suggests a mechanistic relationship to the papain/cathepsin proteases and should facilitate the design of new antiinfective agents.

17p (p53) allelic losses, 4N (G2/tetraploid) populations, and progression to aneuploidy in Barrett's esophagus.

Increased 4N (G2/tetraploid) cell populations have been postulated to be genetically unstable intermediates in the progression to many cancers, but the mechanism by which they develop and their relationship to instability have been difficult to investigate in humans in vivo. Barrett's esophagus is an excellent model system in which to investigate the order in which genetic and cell cycle abnormalities develop relative to each other during human neoplastic progression. Neoplastic progression in Barrett's esophagus is characterized by inactivation of the p53 gene, the development of increased 4N (G2/tetraploid) cell fractions, and the appearance of aneuploid cell populations. We investigated the hypothesis that patients whose biopsies have increased 4N (G2/tetraploid) cell fractions are predisposed to progression to aneuploidy and determined the relationship between inactivation of p53 and the development of 4N abnormalities in Barrett's epithelium. Our results indicate that increased 4N (G2/tetraploid) populations predict progression to aneuploidy and that the development of 4N abnormalities is interdependent with inactivation of the p53 gene in Barrett's esophagus in vivo.

Olfactory neuroblastoma is a peripheral primitive neuroectodermal tumor related to Ewing sarcoma.

Olfactory neuroblastoma (ONB) is a malignant tumor of the nasal mucosa whose histogenesis is unclear. A relationship to neuroblastoma (NB), a pediatric tumor of the sympathetic nervous system, is based on morphologic similarities and the expression of similar neural antigens. However, the clinical presentation of ONB differs from that of NB, and MYCN amplification characteristic of NB is not observed. We have therefore examined the relationship of this malignancy to other classes of neural tumors. In previous studies, two ONB cell lines demonstrated cytogenetic features and patterns of protooncogene expression suggestive of a relationship to the Ewing sarcoma family of childhood peripheral primitive neuroectodermal tumors (pPNETs). The pPNETs show t(11;22)(q24;q12) or t(21;22)(q22;q12) chromosomal translocations fusing the EWS gene from 22q12 with either the FL11 gene on 11q24 or the ERG gene on 21q22. We therefore analyzed ONBs for the presence of pPNET-associated gene fusions. Both cell lines showed rearrangement of the EWS gene, and fluorescence in situ hybridization (FISH) of each case demonstrated fusion of EWS and FL11 genomic sequences. Moreover, both lines expressed EWS/FL11 fusion transcripts with in-frame junctions between exon 7 of EWS and exon 6 of FL11 as described for pPNETs. We identified similar gene fusions in four of six primary ONB cases. None of the cases expressed tyrosine hydroxylase, a catecholamine biosynthetic enzyme widely expressed in NB. Our studies indicate that ONB is not a NB but is a member of the pPNET family.